Project Summary The asymmetric distribution of lipids between the two leaflets of the plasma membrane (PM) bilayer is a fundamental feature of cells across the tree of life. Establishing and maintaining disparate lipid compositions in apposing leaflets is energetically costly, implying an important physiological role for membrane asymmetry. Classically, the lipid asymmetry has been considered largely in the context of apoptosis, where the exposure of inner leaflet lipids on the cell surface marks dead cells for macrophagic engulfment. However, the non- uniform transbilayer lipid distribution of the PM is also involved in a number of other cellular contexts. For example, concentrating anionic lipids on the inner PM leaflet produces a high surface charge density and recruits positively charged proteins. Furthermore, it has become evident that a reversible loss of lipid asymmetry also occurs during healthy cell signaling, most notably in antigen-stimulated activation of a variety of immune cell types. These insights reveal a central, yet poorly understood role for both steady-state membrane asymmetry and its transient loss in immune signaling; however, they also highlight major knowledge gaps in our understanding of PM asymmetry. Specifically, the compositions and biophysical properties of the two leaflets of the PM bilayer are currently unknown, as are their changes during scrambling, either induced by apoptosis or healthy cell signaling. Finally, how asymmetry contributes to immune cell activation is almost completely not understood. We have developed a novel set of methodologies that enable us to address these questions of fundamental importance to cell biology in general, and immune signaling in particular. Based on extensive preliminary data, we propose that immune cells transiently scramble PM lipids during antigen-mediated activation to regulate charge-dependent interactions of signaling proteins with the PM. In Aim 1, we will define the changes in lipidomic and biophysical asymmetry occurring in immune cell PMs following activation by specific antigens. These changes will be compared to the robust PM scrambling induced by apoptotic stimuli. We will also probe the molecular mediators of these effects in both mast cells and T-cells. In Aim 2, we assess the functional consequences of antigen-induced PM scrambling in immune cells, and the molecular mechanisms underlying these effects. First, we will define the role of PM scrambling in immune cell activation by measuring how various functional phenotypes (including cytokine secretion, degranulation, and signaling protein activation) are affected by inhibition or knock-out of PM scrambling machinery. We will then probe the mechanistic connections between PM scrambling and immune activation by cellular, model membrane, and in silico investigations of protein-membrane interactions probing the effect of PM charge density on association of polybasic proteins with the PM. This detailed, comprehensive characterization of the compositional, biophysical, and functional asymmetry of immune cell PMs will elucidate central organizational principles in mammalian cells and how these contribute to immune physiology.